Protein isoform discrimination and quantitative measurements thereof

Abstract

The invention relates to methods, reagents and apparatus for detecting protein isoforms (e.g., those due to alternative splicing, or different disease protein isoforms or degradation products) in a sample, including using combinations of capture agents to identify the isoforms to be detected / measured.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application is a continuation-in-part of U.S. patent application Ser. No. 11 / 387,389, filed Mar. 23, 2006, the entire disclosure of which is incorporated by reference herein for all purposes.BACKGROUND OF THE INVENTION [0002] Antibody arrays are useful for detecting multiple proteins simultaneously in a biological sample. An important and growing application area for the study of multiple proteins is the identification and quantitative measurement of proteins having similar amino acid sequences, for example, protein isoforms resulting from gene-level events, for example, gene splicing and genetic translocations. [0003] U.S. patent application publication number US 2004-0029292 A1 describes technology that can be used to identify multiple proteins using an array. Briefly, starting from the primary amino acid sequence of any protein, one can identify a series of linear epitopes (PETs) that uniquely represent the protein. By de...

AI Technical Summary

Benefits of technology

[0124] An important advantage of the invention is that useful capture agents can be identified and / or synthesized even in the absence of a sample of the protein to be detected. With the completion of the whole genome in a number of organisms, such as human, fly (e.g., Drosophila melanogaster) and nematode (e.g., C. elegans), PETs of a given length or combinations thereof can be identified for any single given protein in a certain organism, and capture agents for any of these proteins of interest can then be made without ever cloning and expressing the full length protein.

[0125] In addition, the suitability of any PET to serve as an antigen or target of a capture agent can be further checked against other available information. For example, since amino acid sequence of many proteins can now be inferred from available genomic data, sequence from the structure of the proteins unique to the sample can be determined by computer aided searching, and the location of the peptide in the protein, and whether it will be accessible in the intact protein, can be determined. Once a suitable PET peptide is found, it can be synthesized using known techniques. With a sample of the PET in hand, an agent that interacts with the peptide such as an antibody or peptidic binder, can be raised against it or panned from a library. In this situation, care must be taken to assure that any chosen fragmentation protocol for the sample does not restrict the protein in a way that destroys or masks the PET. This can be determined theoretically and / or experimentally, and the process can be repeated until the selected PET is reliably retrieved by a capture agent(s).

[0126] The PET set selected according to the teachings of the present invention can be used to generate peptides either through enzymatic cleavage of the protein from which they were generated and selection of peptides, or preferably through peptide synthesis methods.

[0127] Proteolytically cleaved peptides can be separated by chromatographic or electrophoretic procedures and purified and renatured via well known prior art methods.

[0128] Synthetic peptides can be prepared by classical methods known in the art, for example, by using standard solid phase techniques. The standard methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis, and even by recombinant DNA technology. See, e.g., Merrifield, J. Am. Chem. Soc., 85:2149 (1963). Solid phase peptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

[0129] Synthetic peptides can be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.] and the composition of which can be confirmed via amino acid sequencing.

Problems solved by technology

However, mammalian genes can undergo modifications that yield modified protein forms with similar amino acid sequences, and only limited unique regions.

Since one or more distinct proteins with similar amino acid sequences may be present in the same protein sample to be analyzed, discrimination of these proteins is a challenging application.

Unfortunately, the present methods for analyzing distinct proteins with similar amino acid sequences in a sample are inadequate.

However, the choice of sequences for raising antibodies that recognize the uniqueness at the junction region is limited based on the amino acids comprising the junction region, and such limited choices may not even be desirable.

For example, the junction region may be too hydrophobic, too short, etc., making such regions poor candidates for raising effective capture agents (e.g., antibodies or functional fragments thereof).

Consequently, it is not always possible to develop antibodies to the junction region.

Further, while sequences at the junction region are generally unique relative to the isoform family, they may not be unique across the entire proteome.

In fact, they may not even be unique for all the other proteins in a given sample to be analyzed.

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